Monitoring of intracellular Calcium ion levels in mice with Apoaequorin transgene
Gene encoding Apoaequorin, the apoprotein of the calcium binding protein, Aequorin, was inserted in the downstream of neuron specific enolase promoter sequence, and transgenic mice carrying Apoaequorin gene was obtained. Apoaequorin gene was found in DNA level. However, the gene expression was considered to be low, since the calcium-dose dependent illuminance was not detected. We are trying to enhance the gene expression by enhancing the promotor activity.
Monitoring of intracellular Calcium ion in stable colony expressing Apoaequorin
Transfection vector PRK7 was constructed by inserting Apoaequorin gene in the downstream of CMV promoter. NIH 3T3 cells were transfected with the vector and the colony which stably expressing Apoaequorin was obtained. The stable colony showed calcium-dose dependent bioluminescence at the calcium levels between 10^<-5> M and 10^<-1> M.Strong flush-like bioluminescence was also detected by applying KC1. Stimulus-dependent increase in the intracellular calcium levels was detected, while the basal resting levels was below the lower limit our photomultipulier. We also measured the luminescence using recombinant aequorin protein. The linear regression curve was obtained fro 20 ng/mu1. Thus, by measuring bioluminescence, aequorin concentration could be estimated and the calcium level could also be estimated, since one molecule of aequorin binds two molecules of calcium ion. This transfection method can be applied to any mammalian cell lines. And real-time monitoring can be continued as iong as the substrate coelenterazin was supplied.